Bacterial metabolism can occur through various different mechanisms depending on the bacterial species and environmental conditions. Heterotrophic bacteria, which include all pathogens, obtain energy from oxidation of organic compounds, with carbohydrates (particularly glucose), lipids and protein being the most commonly oxidised compounds. Biological oxidation of these organic compounds by bacteria results in synthesis of ATP, a chemical energy source. The process also permits generation of more simple organic compounds (precursor molecules) which are required by the bacterial cell for biosynthetic reactions.
Starch is a naturally abundant carbohydrate and is the principal glucose storage complex in plants. Starch molecules consist of two polysaccharides called amylose and amylopectin. Amylose is a linear polymer of 500 to 20,000 D-glucose subunits, which are linked together via α-1,4 glucosidic bonds to form a helical structure. The additional presence of α-1,6 glucosidic bonds results in amylopectin, which has a branched structure. Starch generally comprises 20-30% amylose and 70-80% amylopectin. In plant cells, insoluble starch is packed into solid granules in which amylopectin is clustered in crystalline regions and amylose is distributed throughout. Starch solubility increases with temperature; amylopectin crystals become gelatinous and granules eventually dissolve.
Amylase is a calcium-dependant glycoside hydrolase metalloenzyme. There are three forms of amylase (α, β and γ) which vary according to the specific bonds they hydrolyse. Alpha-amylase catalyses the random hydrolysis of internal α-D-1,4 glucosidic bonds, releasing simple fermentation sugars including glucose, maltose (disaccharides formed by two glucose units). Dextrins (short, low molecular weight α-1,4-linked D-glucose polymers) are released by amylopectin hydrolysis and maltotriose and maltose are released by amylase hydrolysis. Beta-amylase acts from the non-reducing end of the starch chain, catalysing the hydrolysis of the second α-1,4 glucosidic bond to cleave two glucose units (maltose). Gamma-amylase has the capacity to cleave α-1,6 linkages in amylopectin. Alpha-amylase is widely synthesized in nature since many organisms can digest starch. In human physiology α-amylase is most prominent in saliva and pancreatic secretions. Microbial α-amylases are classified as either liquefying (randomly cleaves the polysaccharide to form shorter chains) or saccharifying (produces mono-, di-, or trisaccharide units), depending on the points of hydrolysis of the glucose polymer chain.
The general process by which bacteria metabolise suitable substrates is glycolysis, which is a sequence of reactions that converts glucose into pyruvate with the generation of ATP. The fate of pyruvate in the generation of metabolic energy varies depending on the micro-organism and the environmental conditions. The four principal reactions of pyruvate are illustrated in FIG. 1.
First, under aerobic conditions, many micro-organisms will generate energy using the citric acid cycle and the conversion of pyruvate into acetyl coenzyme A, catalysed by pyruvate dehydrogenase (PDH).
Second, under anaerobic conditions, certain ethanologenic organisms can carry out alcoholic fermentation by the decarboxylation of pyruvate into acetaldehyde, catalysed by pyruvate decarboxylase (PDC) and the subsequent reduction of acetaldehyde into ethanol by NADH, catalysed by alcohol dehydrogenase (ADH).
A third reaction, which also occurs in anaerobic conditions, is the conversion of pyruvate to acetyl CoA, catalysed by pyruvate formate lyase (PFL). Acetyl CoA is subsequently converted into acetaldehyde by the enzyme acetaldehyde dehydrogenase (AcDH) and ethanol is produced by the reduction of acetaldehyde catalysed by ADH.
A fourth process is the conversion of pyruvate into lactate which occurs through catalysis by lactate dehydrogenase (LDH).
There has been much interest in using micro-organisms for the production of ethanol using either micro-organisms that undergo anaerobic fermentation naturally or through the use of recombinant micro-organisms which incorporate the pyruvate decarboxylase and alcohol dehydrogenase genes. The use of such micro-organisms, modified to enhance utilisation of starch as a metabolic substrate, would enable efficient production of ethanol from cheap, abundant, un-refined plant material.
Thermophilic bacteria have been proposed for ethanol production, and their use has the advantage that fermentation can be carried out at elevated temperatures which allows the possibility that the ethanol produced can be removed as vapour at temperatures above 50° C.; this also permits fermentation to be carried out using high substrate concentrations. However, there is a need for improved micro-organisms for ethanol production from starch-based culture media.